Post-translational modifications (PTMs) regulate biological outcomes by influencing protein structure, localization, function, and interactions. While numerous PTMs have been detected to date, often the dynamics and role of these PTMs in specific biological contexts remains unclear. This dissertation highlights my work interrogating the dynamics and role of two PTMs: acetylation on a proteome-wide scale, and methylation on lysine 27 of the histone variant H3.3 in the context of stem cell differentiation. We performed a comprehensive characterization of protein acetylation dynamics using mass spectrometry based proteomics through utilization of 13C-glucose or D3-acetate, which are metabolized into acetyl-CoA, labeling acetyl groups through subsequent incorporation into proteins. We characterized around 1,000 sites with significantly increasing acetylation trends. Faster rates were enriched on proteins associated with chromatin and RNA metabolism, while slower rates were more typical on proteins involved with lipid metabolism. We identified sites catalyzed at faster rates with potential critical roles in protein activation, including the histone acetyltransferase p300 acetylated in its activation loop. This study highlights the dynamic nature of protein acetylation, and how metabolism plays a central role in this regulation. Histone variants, such as histone H3.3, replace canonical histones within the nucleosome to alter chromatin accessibility and gene expression. We demonstrate through methylation dynamics studies that methylation on H3.3K27 is maintained more than on canonical H3K27 over stem cell differentiation. Using a custom-made antibody, we identify a distinct enrichment of H3.3K27me3 at lineage-specific genes, such as olfactory receptor genes, and at binding motifs for the transcription factors FOXJ2/3. REST, a predicted FOXJ2/3 target that acts as a transcriptional repressor of terminal neuronal genes, was identified with H3.3K27me3 at its promoter region. H3.3K27A mutant cells confirmed an upregulation of FOXJ2/3 targets upon the loss of methylation at H3.3K27. Thus, while canonical H3K27me3 has been characterized to regulate the expression of transcription factors that play a general role in differentiation, our work suggests H3.3K27me3 is essential for regulating distinct terminal differentiation genes. This work highlights the importance of understanding the effects of PTMs not only on canonical histones but also on specific histone variants, as they may exhibit distinct roles.